Theexpression And Biological Significance Of MiR-506 In Malignant Melanoma

Objective:Malignant melanoma(MM) is one of the most deadly tumors and primarily derives from melanocytes with a poor prognosis in advanced stage. The median overall survival time of metastatic MM was only 7.5 months. However,the incidence of MM has been ascending stably for years in China, accompanied by increasing mortality. Traditional therapies(chemotherapy and radiotherapy) are not effective enough in managing metastatic MM patients. Fortunately, the targeted therapeutic drugs andimmunotherapy such as Vemurafenib and Ipilimumab haveshown their special advantage in the treatment of advanced MM. Micro RNAs(miRNAs) are a group of short non-coding RNAs of ~22 nucleotides inlength and could modulate at least 30% human genes` expression by targeting multiple m RNAs, acting as oncogenes or anti-oncogenes in tumor. Recent studies have identified that miR-506 serves as tumor suppressor in ovarian cancer, breast cancer, gastric cancer and some other epithelial cancers, but the role of miR-506 in MM is unclear. In this study, we aim at determining the expression and significance of miR-506 in melanoma, exploring the regulatory mechanism of miR-506 in mesenchymal-epithelial transition(MET)and DNA homologous recombination signal pathway, and identifying the biological significance of miR-506 in MM. Methods:This study recruited 776 cases of MM confirmed by pathology from February 1981 to May 2013 at Tianjin Medical University Cancer Institute and Hospital. The clinical and pathological characteristics were obtained by epidemiology survey.Real-time quantitative PCR was used to examine the relative expression of miR-506 in 48 MM tissues(16 paired tumor and normal tissues).We selected 147 cases from the total 776 cases with formalin fixed paraffin embedded(FFPE)tumor tissues and made into tissue microarray(TMA). Immunohistochemistry was used to assess the expression of DNA damage homologous recombination related protein(RAD51 recombinase, RAD51; RAD52 homolog, DNA repair protein, RAD52; ATM serine/threonine kinase, ATM) and MET related protein(N-cadherin, Vimentin,Slug and E-cadherin)and then made the correlation analysis of protein expression in each group.The relationship between the expression of miR-506, related proteins and the clinicopathological features were explored by Chi-square test. Survival analysis(including overall survival and disease-free survival) was calculated by Kaplan-Meier method.Overexpress or inhibit miR-506 in MM cell lines A375 and A875, and then we used 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT), transwell chamber assay to observe the effect of miR-506 on cell proliferation, migration and invasion in MM. Western blot assay was used to test the effect of miR-506 on the expression of MET related protein(N-cadherin, Slug and Vimentin)and DNA homologous recombination related protein(RAD51).Results: 1. Real-time quantitative PCR indicated that the relative expression of miR-506 was significantly upregulated in melanoma tissue than non-tumor tissue(n=64,P=0.006). Higher expression of miR-506 had better overall survival(OS), disease-free survival(DFS) and progress-free survival(PFS) in melanoma patients(OS: n=64, P=0.002, DFS: n=32, P<0.001, PFS, n=35, P=0.039, respectively). The expression of miR-506 in metastatic melanoma was significantly lower than primary melanoma(P=0.049).2. The positive expression of RAD51 and ATM was 56.5% and 53.7%, respectively. The expression of RAD51 and ATM had a positive correlation(r=0.396,P<0.001), however, the expression of RAD52 was inversely related to RAD51 and ATM(r=-0.253, P=0.002; r=-0.219, P=0.008), respectively. Additionally, the expression of miR-506 was negatively associated with the expression of RAD51(r=-0.467, P=0.001).3. The positive expression of Vimentin, N-cadherin, Slug and E-cadherin were 72.1%, 36.1%, 40.1% and 29.3%,respectively. Moreover, the association between N-cadherin and E-cadherin was negative(r=-0.327, P<0.001)and positive with Vimentin and Slug( r=0.246, P=0.003; r=0.281, P=0.001). Furthermore, Vimentin and Slug also had a positive correlation(r=0.385, P<0.001).And the expression of miR-506 was negatively associated with the expression of Vimentin and Slug(r=-0.377, P=0.008; r=-0.358, P=0.012).4. RAD51, ATM and N-cadherin were worse prognosticators of melanoma patients` survival, but RAD52 and E-cadherin was indicated as the opposite predictor. What is more, multivariate analysis indicated that RAD51 ? RAD52 and E-cadherin could be the independent prognostic factors of melanoma patients` OS(P=0.025, HR=2.41; P=0.008, HR=0.37; P=0.003, HR=0.21).5. MTT and Transwell chamber experiment in melanoma cell A375 and A875 indicated that overexpression of miR-506 could inhibit the function of cellproliferation, migration and invasion the inhibition of miR-506 could increase cell proliferation, migration and invasion.6. Increase the expression of miR-506 in melanoma cellscould significantly downregulate Vimentin, N-cadherin, Slug and RAD51.7. Suppressthe expression of miR-506 in melanoma cells could not upregulate Vimentin, N-cadherin, Slug and RAD51.Conclusion The expression of miR-506 is closely associated with the prognosis of melanoma. Overexpression of miR-506 coulddownregulate the expression of RAD51, Vimentin, N-cadherin and Slug, and may promote the process of Mesenchymal-Epithelial Transition(MET) and DNA homologous combination, as well as suppress the proliferation, migration and invasion in melanoma cells. The above results indicate the miR-506 may act as aprotect factor in melanoma.